ORIGINAL ARTICLE Molecular Signature of Tumors with Monoallelic 13q14 Deletion: a Case Series of Spindle Cell Lipoma and Genetically-Related Tumors Demonstrating a Link Between FOXO1 Status and p38 MAPK Pathway Karina Uehara 1 & Fukino Ikehara 1 & Ryo Shibuya 2 & Iwao Nakazato 3 & Mariko Oshiro 4 & Masaya Kiyuna 5 & Yasuka Tanabe 1 & Zensei Toyoda 1 & Kiyoto Kurima 6 & Shinichiro Kina 7 & Masanori Hisaoka 2 & Takao Kinjo 1 Received: 23 May 2017 /Accepted: 1 September 2017 /Published online: 8 September 2017 # The Author(s) 2017. This article is an open access publication Abstract Spindle cell/pleomorphic lipomas (SCLs), cellu- lar angiofibromas (CAFs) and mammary-type myofibroblastomas (MFBs) are rare benign mesenchymal tumors with monoallelic 13q14 deletion. They are predict- ed to have a common pathogenic mechanism due to shared similar histological and immunohistochemical fea- tures; however, pathological consequences of monoallelic 13q14 deletion remain unknown. We previously reported a CAF case with monoallelic 13q14 deletion in which the tumor expressed decreased levels of FOXO1 and RB1, both of which were encoded in 13q14, and increased reactive oxygen species (ROS) levels. We further demon- strated the activation of p38 mitogen-activated protein ki- nase (p38 MAPK) pathway induced by oxidative stress. We hypothesized that SCLs, CAFs and MFBs would share common molecular signatures involving FOXO1, ROS and p38 MAPK and that their expression patterns were different from those tumors without monoallelic 13q14 deletion such as solitary fibrous tumors (SFTs). We compared the expression levels of FOXO1, RB1, ROS markers and several signal transduction factors be- tween SCLs and SFTs. SCLs expressed decreased levels of FOXO1 and RB1, whereas SFTs showed no change. Both tumor types exhibited increased markers of ROS; however, nuclear localization of phosphorylated p38 was significantly more frequent in SCLs than that in SFTs, suggesting p38 MAPK activation by oxidative stress. SFTs showed lower p38 MAPK activity and higher β- catenin expression, implying that oxidative stress was caused by increased cellular proliferation stress. Finally, CAFs and MFBs showed changes similar to those ob- served in SCLs. Overall, tumors with monoallelic 13q14 deletion showed shared molecular signatures that might be associated with pathogenesis. Keywords Monoallelic 13q14 deletion . RB 1 . FOXO 1 . Reactive oxygen species . p38 MAPK Abbreviations SCL Spindle cell/pleomorphic lipoma CAF Cellular angiofibroma MFB Mammary-type myofibroblastoma SFT Solitary fibrous tumor * Takao Kinjo [email protected]1 Division of Morphological Pathology, Department of Basic Laboratory Sciences, School of Health Sciences, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan 2 Department of Pathology and Oncology, School of Medicine, University of Occupational and Environmental Health, Fukuoka, Japan 3 Department of Pathology, Okinawa Prefectural Nanbu Medical Center and Children’ s Medical Center, Okinawa, Japan 4 Health Information Management Major, Management and Information Science Division, Faculty of International Studies, Meio University, Okinawa, Japan 5 Department of Pathology, Tomishiro Chuo Hospital, Okinawa, Japan 6 Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan 7 Department of Oral and Maxillofacial Functional Rehabilitation, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan Pathol. Oncol. Res. (2018) 24:861–869 DOI 10.1007/s12253-017-0303-6
Transcript
ORIGINAL ARTICLE
Molecular Signature of Tumors with Monoallelic 13q14 Deletion:a Case Series of Spindle Cell Lipoma and Genetically-RelatedTumors Demonstrating a Link Between FOXO1 Status and p38MAPK Pathway
Received: 23 May 2017 /Accepted: 1 September 2017 /Published online: 8 September 2017# The Author(s) 2017. This article is an open access publication
Abstract Spindle cell/pleomorphic lipomas (SCLs), cellu-l a r ang io f i b romas (CAFs ) and mammary - t ypemyofibroblastomas (MFBs) are rare benign mesenchymaltumors with monoallelic 13q14 deletion. They are predict-ed to have a common pathogenic mechanism due toshared similar histological and immunohistochemical fea-tures; however, pathological consequences of monoallelic13q14 deletion remain unknown. We previously reporteda CAF case with monoallelic 13q14 deletion in which thetumor expressed decreased levels of FOXO1 and RB1,both of which were encoded in 13q14, and increased
reactive oxygen species (ROS) levels. We further demon-strated the activation of p38 mitogen-activated protein ki-nase (p38 MAPK) pathway induced by oxidative stress.We hypothesized that SCLs, CAFs and MFBs wouldshare common molecular signatures involving FOXO1,ROS and p38 MAPK and that their expression patternswere different from those tumors without monoallelic13q14 deletion such as solitary fibrous tumors (SFTs).We compared the expression levels of FOXO1, RB1,ROS markers and several signal transduction factors be-tween SCLs and SFTs. SCLs expressed decreased levelsof FOXO1 and RB1, whereas SFTs showed no change.Both tumor types exhibited increased markers of ROS;however, nuclear localization of phosphorylated p38 wassignificantly more frequent in SCLs than that in SFTs,suggesting p38 MAPK activation by oxidative stress.SFTs showed lower p38 MAPK activity and higher β-catenin expression, implying that oxidative stress wascaused by increased cellular proliferation stress. Finally,CAFs and MFBs showed changes similar to those ob-served in SCLs. Overall, tumors with monoallelic 13q14deletion showed shared molecular signatures that mightbe associated with pathogenesis.
1 Division of Morphological Pathology, Department of BasicLaboratory Sciences, School of Health Sciences, University of theRyukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan
2 Department of Pathology and Oncology, School of Medicine,University of Occupational and Environmental Health,Fukuoka, Japan
3 Department of Pathology, Okinawa Prefectural Nanbu MedicalCenter and Children’s Medical Center, Okinawa, Japan
4 Health Information Management Major, Management andInformation Science Division, Faculty of International Studies, MeioUniversity, Okinawa, Japan
5 Department of Pathology, Tomishiro Chuo Hospital, Okinawa, Japan6 Department of Regenerative Medicine, Graduate School of
Medicine, University of the Ryukyus, Okinawa, Japan7 Department of Oral and Maxillofacial Functional Rehabilitation,
Graduate School of Medicine, University of the Ryukyus,Okinawa, Japan
Spindle cell/pleomorphic lipomas (SCLs), cellular angiofibromas(CAFs) and mammary-type myofibroblastomas (MFBs) are rarebenign mesenchymal tumors with monoallelic 13q14 deletion[1–3]. These tumors share similar histopathological and immu-nohistochemical features; however, molecular events induced bythis genetic alteration remain unclear. Two tumor suppressorgenes, RB1 and FOXO1, are located in 13q14. While RB1 is awell-established tumor suppressor, FOXO family of transcriptionfactors are associated with diverse functions such as cell cycleregulation, differentiation, apoptosis, DNA repair and reactiveoxygen species (ROS) detoxification [4–6]. Specifically,FOXO1 was shown to be associated with alveolar rhabdomyo-sarcoma as a result of chromosome alteration and is consideredas a tumor suppressor [4].
In a previous study, we reported a CAF case withmonoallelic 13q14 deletion. The tumor expressed de-creased levels of FOXO1 and RB1 as well as increasedlevels of oxidative stress markers, 8-hydroxy-2 ′-deoxyguanosine (8-OHdG) and 4-hydroxy-2-nonenal (4-HNE). Furthermore, the p38 mitogen-activated protein ki-nase (p38 MAPK) pathway, which is often induced byROS, was activated in that CAF case [7].
While the molecular mechanism underlying tumorigen-esis associated with monoallelic 13q14 deletion is notknown, based on these findings, we hypothesized thatSCLs, CAFs and MFBs would share common molecularsignatures involving FOXO1 expression, ROS status andp38 MAPK activity. To examine our hypothesis, we con-ducted a series of comparative studies between tumorswith monoallelic 13q14 deletion, SCLs, CAFs andMFBs, and those without 13q14 deletion, such as solitaryfibrous tumors (SFTs), aggressive angiomyxomas (AAs),dermatofibrosarcoma protuberans (DFSP), well-differentiated liposarcoma (WLS), myxoid liposarcoma(MLS) and pleomorphic liposarcoma (PLS) which are fre-quently considered in differential diagnosis. We analyzedclinicopathological characteristics and performed immu-nohistochemistry to assess the expression levels ofFOXO1, RB1 and oxidative stress markers, the p38MAPK activity and other signal transduction pathwaysassociated with pathogenesis.
Methods
Case Selection
Representative hematoxylin and eosin-stained slides ofcases were reviewed for case selection. The majority ofcases were derived from the consultation files of one ofthe authors (M. H.), which included 52 SCLs, 3 CAFs, 3MFBs, 36 SFTs and 2 AAs. In addition, 3 SCLs, 1 CAF, 4SFTs, 2 AAs, 11 DFSP, 12 WLS, 10 MLS and 4 PLSwere retrieved from the surgical pathology files of otherinstitutions. Several aspects of clinical, histological, im-munohistochemical and cytogenetic information on aCAF sample were previously reported [7]. Since theSFT harbor inversion of chromosome 12q13 resulting inNAB2-STAT6 fusion gene, detection of STAT6 by immu-nohistochemistry has become a diagnostic tool for SFT. Inpresent study, 91% (32/35) of SFT were positive forSTAT6. The study protocol was approved by theInstitutional Review Boards of University of theRyukyus, Okinawa, Japan (No. 791, June 2, 2015) andUniversity of Occupational and Environmental Health,Fukuoka, Japan (H27-029, June 23, 2015). All proceduresperformed in this study were carried out in accordancewith the Declaration of Helsinki.
Histopathological Analysis
The tumors were processed, stained with hematoxylin andeosin and were analyzed using immunohistochemistry.Primary antibodies used in this study were α-smooth muscleactin (Dako, Glostrup, Denmark), CD34 (Dako), desmin(Dako), Ki-67 (Dako), S-100 (Dako), RB1 (Abcam,Cambridge, United Kingdom), p16 (Abcam), STAT6 (SantaCruz, Dallas, Texas), FOXO1 (Cell Signaling Technology,Boston, Massachusetts), phosphorylated p38 (Cell SignalingTechnology), β-catenin (Millipore, Billerica, Massachusetts),8-OHdG or 4-HNE (both from Japan Institute for the Controlof Aging, Shizuoka, Japan), and the details of the stainingmethods were described in the previous report [7]. The appro-priate positive and negative controls were included in eachstaining. The immunoreactivity and positive extent of themarkers were semiquantitatively scored using a similar meth-od as that reported by Sangoi et al. [8]. In brief, the immuno-reactivity was graded as negative, weak, moderate, or strong.The percentage of tumor cells with each marker was semi-quantitatively scored as 0 (0%–9%), 1+ (10%–49%), 2+(50%–89%) and 3+ (≥90%). For each antibody, the immuno-reactivity of more than weak and the percentage score of tu-mor cells more than 1+ were judged as positive. Only nuclearpositive signals for RB1, FOXO1, 8-OHdG, p16, phosphory-lated p38 and β-catenin were used for scoring.
862 K. Uehara et al.
Fluorescence In Situ Hybridization Analysis
Fluorescence in situ hybridization (FISH) analysis was per-formed using a chromosome 13 (13q14)-specific probe(Poseidon™ repeat-free FISH probes, Kreatech Diagnostics,Amsterdam, The Netherlands), which include RB1 gene butnot FOXO1 gene, according to themanufacturer’s instructionsand as described previously [7].
Statistical Analysis
Data were analyzed by two-tail Mann-Whitney U-test. A Pvalue of <0.05 was considered statistically significant for allanalyses.
Results
Clinical Findings of Tumors with Monoallelic 13q14Deletion
The summary of clinicopathological parameters of tumorswith monoallelic 13q14 are shown in Table 1. The SCLs af-fected 45 males and 8 females; two cases were unknown. Themedian age was 62 years (range, 21–87 years). The mostcommon affected sites were the head and neck, followed bythe trunk. The CAFs affected 3 males and 1 female, and themedian age was 53.5 years (range, 49–69 years). The CAFfrom female was in the vagina, whereas the CAFs from themales were in the groin, testis and scrotum. The MFBs affect-ed 1 male and 2 females, with a median age of 64 years (range,54–65 years). The MFBs from the females were in the groinand knee, whereas the MFB from the male was in the mam-mary gland.
FISH for 13q14
All SCLs were examined by FISH for 13q14. Among atotal of 55 SCLs, monoallelic 13q14 deletion was con-firmed in 43 cases (78%), and 12 cases unsuitable forFISH or negative for monoallelic 13q14 deletion wereexcluded for subsequent analyses.
RB1 and FOXO1 Expression in Tumors with orWithout Monoallelic 13q14 Deletion
RB1 and FOXO1 expression was evaluated because thesegenes are encoded in 13q14 region. Nuclear RB1 expressionwas confirmed in 29 of 42 (69%) SCLs; however, the majorityexhibited either weak or moderate immunoreactivity. RB1expression was also detected in 1 of 4 CAFs and all MFBs(3/3). SFTs showed higher rates of positive RB1 expression(32/40; 80%) than those observed in SCLs, and the
immunoreactivity to RB1 in SFTs tended to be higher thanthat observed in SCLs. Finally, PLSs (0/4) showed no expres-sion and WLSs (3/11) demonstrated low frequency, whereasthe others including AAs (3/4), DFSPs (5/11) and MLSs(8/10) were positive for RB1 (Table 2, Fig. 1).
Nuclear FOXO1 expression was detected in 16 of 30(53%) SCLs. The majority of FOXO1-positive cases exhibit-ed weak immunoreactivity. FOXO1 was expressed in 1 of 4CAFs, and in all the MMF specimens analyzed; however,none of those showed a strong FOXO1 immunoreactivity.The majority of SFTs (31/36: 86%) were positive forFOXO1, and FOXO1 immunoreactivity tended to be higherthan that observed in SCLs. Although PLSs (0/4) showed noexpression, AAs (2/4), DFSPs (5/11), WLSs (9/11) and MLSs(9/10) expressed FOXO1 (Table 2, Fig. 1).
Oxidative Stress Markers in Tumors with orWithout Monoallelic 13q14 Deletion
Two oxidative stress markers were evaluated in this study. Themajority of SCLs were positive for both 8-OHdG (36/42:86%) and 4-HNE (34/42: 81%). The majority of CAFs andMFBs also exhibited positive staining for these two markers.Similarly, SFTs also showed increased expression of 8-OHdG(31/40: 78%) and 4-HNE (34/40: 85%). Finally, most of theAAs, DFSPs, WLSs, MLSs and PLSs indicated high positiv-ity for both markers (Table 2, Fig. 2).
p38 MAPK and β-Catenin Signaling in Tumors with orWithout Monoallelic 13q14 Deletion
Nuclear phosphorylated p38 localization was demonstrated in28 of 42 (67%) SCLs. The majority of those positive for phos-phorylated p38 showed moderate immunoreactivity. A posi-tive nuclear phosphorylated p38 was also seen in all 4 CAFs,as well as 2 out of 3 MMFs. Of a total 40 SFTs, 15 casesshowed nuclear phosphorylated p38 localization (15/40;38%); however, the extent and immunoreactivity of phosphor-ylated p38 in SFTs tended to be lower than those observed inSCLs. Finally, most of AAs and MLSs were positive for nu-clear phosphorylated p38, whereas DFSPs, WLSs and PLSsshowed low positive rate (Table 2, Fig. 3).
Because p16 is one of the important signal transductionfactors downstream from p38 MAPK pathway, we comparedexpression of p16 between SCLs and SFTs. All SCLsexpressed p16 (42/42: 100%), and among them, 38 cases wereeither moderately or strongly immunoreactive for p16. OfSFTs, 76% (29/38) were positive for p16, whereas p16 immu-noreactivity varied across specimens (Table 2, Fig. 3).
Nuclear β-catenin localization was observed in 15 of 42(36%) SCLs; the β-catenin immunoreactivity was either mod-erate or weak in these cases. No nuclear β-catenin was detect-ed in CAFs, whereas 1 out of 3 MMFs expressed nuclear β-
Molecular Signature of Tumors with Monoallelic 13q14 Deletion: a Case Series of Spindle Cell Lipoma and... 863
catenin. In contrast, the majority of SFTs (28/40: 70%) werepositive for β-catenin which tended to be expressed strongerthan that observed in SCLs (Table 2, Fig. 3).
Discussion
SCLs, CAFs and MFBs share a common genetic defect:monoallelic 13q14 deletion [1–3]. While two tumor sup-pressor genes, RB1 and FOXO1, are encoded in thisregion, the precise molecular pathogenesis of these tu-mors is unknown. To date, there are relatively few re-ports investigating the pathogenesis of these tumors.Chen et al. reported that soft tissue tumors withmonoallelic 13q deletions, such as SCLs and CAFs aswell as the majority of MMFs did not express RB1,which might lead to subsequent tumor development.Although the mechanism underlying the decreased levelof RB1 expression is yet unclear, they suggested a mu-tation on the remaining allele or an epigenetic changethat affects the expression of RB1 [9]. Magro et al. re-ported deletion of FOXO1 in mammary- and vaginal-
type MFBs by FISH analysis and proposed that tumorswith 13q14 deletion belong to a single entity with acontinuous spectrum of different morphological presenta-tions [10]. However, FOXO1 expression level in thesetumors has not been reported. In the present study, wecompared the expression of RB1 and FOXO1 betweenSCLs (tumor with monoallelic 13q14 deletion) andSFTs (that without 13q14 deletion), because the numberof cases is sufficient for statistical analysis between thesetumors. Contrary to our prediction, the rate of RB1 pos-itivity was not different between SCLs and SFTs, how-ever, the extent of positive signal and immunoreactivityto RB1 in SCLs were lesser than those in SFTs. Becausewe used 13q14 FISH probe which included RB1 region,one signal in the present FISH experiment suggested lossof one RB1 gene. Therefore, the extent and immunore-activity of RB1 in SCLs may reflect the gene dosage ofRB1, i.e., one allele of RB1 gene by monoallelic dele-tion. The difference of RB1 positivity between presentstudy and the study by Chen et al. (2012) could havebeen due to the different anti-RB1 antibody used. Thepositive rate as well as both positive extent and
Table 1 Summary ofclinicopathological features ofspecimens analyzed in this study
SCL CAF MFB AA SFT DFSP WLS MLS PLS
Sex
Male 45 3 1 1 24 7 8 5 1
Female 8 1 2 3 16 5 4 5 3
Age(y)
< 50 11 1 0 2 13 8 2 5 0
≧50 42 3 3 2 27 4 10 5 4
Median 62.0 53.5 64.0 44.0 58.5 44 58.5 49.5 66
Range 21–87 49–69 54–65 28–57 9–84 3–68 40–79 33–81 61–92
Molecular Signature of Tumors with Monoallelic 13q14 Deletion: a Case Series of Spindle Cell Lipoma and... 865
immunoreactivity to FOXO1 in SCLs were lower thanthose in SFTs (53% versus 86%, p < 0.05). Becausethe FISH probe used in the present study did not includeFOXO1 region, and FOXO1 gene is approximately7,8 Mb apart from RB1 gene, the monoallelic 13q14deletion did not necessarily demonstrate the loss of oneallele of FOXO1 gene. The expression control ofFOXO1 may have a similar expression mechanism ofFOXP2, one of FOX gene family, and its mutation isassociated with a neurodevelopmental defect in acquisi-tion of spoken language. FOXP2 undergoes randommonoallelic expression (RMAE), which results in threedistinct expression statuses: expression of Bboth alleles^in some cells and expression of either the maternal orpaternal allele in other cells. FOXP2 expression of Bbothalleles^ is controlled under cis-regulatory element thatmap to 3 Mb away from the FOXP2 gene locus.Therefore, a deletion of the cis-regulatory element, whichis demonstrated in a patient with developmental verbaldyspraxia, results in FOXP2 haploinsufficiency(monoallelic expression) [11]. Because cis-regulatory el-ement of FOXO1 in 13q14 is unknown and whetherFOXO1 operates RMAE is yet unclear, further studyregarding the mechanism of FOXO1 expression is re-quired to understand the impact of monoallelic 13q14deletion. The present study suggested that monoallelic13q14 deletion in SCLs affects the expression of bothRB1 and FOXO1. The number of CAFs and MFBs werelimited in our study; however, both CAFs and MFBsshowed low expression levels of RB1 and FOXO1.Based on the downregulation of both these tumor sup-pressors, we hypothesize that these events may be
contributing to a shared pathogenic mechanism of tumorswith monoallelic 13q14 deletions, as proposed by Magroet al. [10].
In a previous study, we reported a CAF patient withmonoallelic 13q14 deletion and loss of FOXO1 expres-sion, which was accompanied by increased expression ofoxidative stress markers [7]. We also showed the activa-tion of p38 MAPK pathway, which is often induced byoxidative stress [12, 13]. The present study confirmed theincreased oxidative stress and subsequent p38 MAPK ac-tivation in SCLs. These findings suggested that the loss ofFOXO1 function in tumors with monoallelic 13q14 dele-tion induces oxidative stress, leading to the activation ofp38 MAPK signaling. The role of p38 MAPK signaling insoft tissue tumors remains controversial. Two groups re-ported that therapy using SRC family kinase inhibitors, orartesunate, antimalarial drug, induced growth inhibition ofrhabdomyosarcoma through the activation of p38 MAPK[14, 15], whereas another group reported that p38 MAPKmight play a role in induction of human telomerase re-verse transcriptase in sarcomas, suggesting an associationbetween p38 MAPK and malignant transformation [16].
In this study, we found higher p38 MAPK activity in SCLsthan that in SFTs (67% versus 38%, p < 0.05). While the rateand intensity of oxidative stress marker expression were sim-ilar between SCLs and SFTs, the responses to oxidative stressmight differ between them. Given the low rate of FOXO1positivity in SCLs, the response to oxidative stress might beimpaired, resulting in increased ROS levels that lead to p38MAPK activation. Since p16 is one of the important signaltransduction factors downstream from p38 MAPK pathway,the high rate of p16 positivity in SCLs also suggested p38
MAPK activation. CAFs and MFBs showed similar results tothose in SCLs. These findings suggested that tumors withmonoallelic 13q14 deletion express decreased FOXO1 levels,leading to increased ROS accumulation that result in the acti-vation of p38 MAPK pathway.
Despite similar levels of ROS between SCLs and SFTs,FOXO1 positivity rate was higher in SFTs than SCLs, sug-gesting that the response to ROS elicited by FOXO1 in SFTsis intact. In addition, p38 MAPK activity was lower in SFTsthan SCLs; again, these results might reflect an intact FOXO1mediated antioxidant response in SFTs. In general, oxidativestress in cancer cells is aggravated by oncogene expression,increased metabolic activity, hypoxic conditions, chronic in-flammation and damaged mitochondrial function [17–19]. Asneither oxidative stress nor p38 MAPK activity in SFTs hasbeen reported to date, a literature search did not result in anyinformation on the antioxidant response in SFT; however, thepresent study suggests that increased oxidative stress in SFTsinduces normal FOXO1 activation.
Hoogeboom et al. reported that binding of FOXO familymembers to β-catenin inhibits β-catenin/T cell factor (TCF)
transcriptional activity, ultimately resulting in suppression ofthe Wnt signaling [20], implicating FOXO as tumor suppres-sor. Therefore, we analyzed nuclear β-catenin translocationand hypothesized that tumor with monoallelic 13q14 deletionwould show increased nuclear β-catenin translocation.However, in contrast to our hypothesis, β-catenin expressionand its nuclear localization were lower in SCLs than SFTs(36% versus 70%). These results implied that normal func-tions of adenomatous polyposis coli (APC) were preserved inSCLs, which lead to proper proteasomal degradation of β-catenin. Based on these results, we proposed that the Wnt/β-catenin pathway is not related to the pathogenesis in tumorswith monoallelic 13q14 deletion.
In the present study, the number of CAF and MFB caseswas few, therefore, the comparison of positivity of each mark-er between these tumors and SCLs showed limited correlation.Furthermore, the present immunohistochemical analysis dem-onstrated that p38MAPK activation was induced by increasedROS, but the resulting biological effects such as cell prolifer-ation, apoptosis and cell senescence remain unclear. Furthermolecular analyses using fresh specimen is needed.
Fig. 2 Representative images of tumor specimens stained forhematoxylin and eosin, immunohistochemically stained for 8-hydroxy-2′-deoxyguanosine (8-OHdG) and 4-hydroxy-2-nonenal (4-HNE) areshown. SCL: spindle cell/pleomorphic lipoma, CAF: cellularangiofibroma, MFB: myofibroblastoma, SFT: solitary fibrous tumor,DFSP: dermatofibrosarcoma protuberans, WLS: well-differentiatedliposarcoma, MLS: myxoid liposarcoma. Specimens from tumors withmonoallelic deletion of 13q14 as well as those without 13q14 deletiondemonstrated increased expression of markers of oxidative stress
Fig. 1 Representative images of tumor specimens stained forhematoxylin and eosin, immunohistochemically stained for RB1 andFOXO1 are shown. SCL: spindle cell/pleomorphic lipoma, CAF: cellularangiofibroma, MFB: myofibroblastoma, SFT: solitary fibrous tumor,DFSP: dermatofibrosarcoma protuberans, WLS: well-differentiatedliposarcoma, MLS: myxoid liposarcoma. SCL and CAF showed de-creased expression of RB1 and FOXO1, whereas other tumors exhibitedvarious expression levels of either marker
Molecular Signature of Tumors with Monoallelic 13q14 Deletion: a Case Series of Spindle Cell Lipoma and... 867
In summary, the tumors with monoallelic 13q14 deletionshow decreased expression level of FOXO1, which is encodedin the 13q14 region, and increased levels of oxidative stressmarkers as well as higher p38 MAPK activity. The above re-sults lead us to hypothesize that those tumors with monoallelic13q14 deletion share molecular signatures involving the in-crease in the oxidative stress markers that are induced by de-creased FOXO1 expression; furthermore, increased oxidativestress result in the activation of p38 MAPK pathway. The pres-ent study suggests these molecular events are associated withtumorigenesis caused by 13q14 deletion.
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References
1. Flucke U, van Krieken JH, Mentzel T (2011) Cellularangiofibroma: analysis of 25 cases emphasizing its relationship tospindle cell lipoma and mammary-type myofibroblastoma. ModPathol 24:82–89
2. Fritchie KJ, Carver P, Sun Y, Batiouchko G, Billings SD, RubinBP, Tubbs RR, Goldblum JR (2012) Solitary fibrous tumor: isthere a molecular relationship with cellular angiofibroma, spin-dle cell lipoma, and mammary-type myofibroblastoma? Am JClin Pathol 137:963–970
3. Hameed M, Clarke K, Amer HZ, Mahmet K, Aisner S (2007)Cellular angiofibroma is genetically similar to spindle cell lipo-ma: a case report. Cancer Genet Cytogenet 177:131–134
4. Greer EL, Brunet A (2005) FOXO transcription factors at theinterface between longevity and tumor suppression. Oncogene24:7410–7425
6. Maiese K, Chong ZZ, Shang YC, Hou J (2009) A BFOXO^ insight: targeting Foxo proteins from conception to cancer. MedRes Rev 29:395–418
7. Arakaki K, Chinen K, Kamiya M, Tanabe Y, Tawata N,Ikehara F, Uehara K, Shimabukuro H, Kinjo T (2014)Evidence for an association between increased oxidative stressand derangement of FOXO1 signaling in tumorigenesis of acellular angiofibroma with monoallelic 13q14: a case report.Int J Clin Exp Pathol 7:8972–8979
8. Sangoi AR, Dulai MS, Beck AH, Brat DJ, Vogel H (2009)Distinguishing chordoid meningiomas from their histologicmimics: an immunohistochemical evaluation. Am J SurgPathol 33:669–681
9. Chen BJ, Marino-Enriquez A, Fletcher CD, Hornick JL (2012)Loss of retinoblastoma protein expression in spindle cell/pleomorphic lipomas and cytogenetically related tumors: an immu-nohistochemical study with diagnostic implications. Am J SurgPathol 36:1119–1128
10. Magro G, Righi A, Casorzo L, Antonietta T, Salvatorelli L,Kacerovska D, Kazakov D, Michal M (2012) Mammary and vag-inal myofibroblastomas are genetically related lesions: fluorescencein situ hybridization analysis shows deletion of 13q14 region. HumPathol 43:1887–1893
11. Adegbola AA, Cox GF, Bradshaw EM, Hafler DA, Gimelbrant A,Chess A (2015) Monoallelic expression of the human FOXP2speech gene. Proc Natl Acad Sci U S A 112:6848–6854
12. Wang X, Liu JZ, Hu JX, Wu H, Li YL, Chen HL, Bai H, Hai CX(2011) ROS-activated p38 MAPK/ERK-Akt cascade plays a cen-tral role in palmitic acid-stimulated hepatocyte proliferation. FreeRadic Biol Med 51:539–551
13. Torres M, Forman HJ (2003) Redox signaling and the MAP kinasepathways. Biofactors 17:287–296
14. Beccafico S, Morozzi G, Marchetti MC, Riccardi C, Sidoni A,Donato R, Sorci G (2015) Artesunate induces ROS- and p38MAPK-mediated apoptosis and counteracts tumor growthin vivo in embryonal rhabdomyosarcoma cells. Carcinogenesis36:1071–1083
Fig. 3 Representative images of tumor specimens immunohistochemicallystained for phosphorylated p38, p16 and β-catenin are shown. SCL: spindlecell/pleomorphic lipoma, CAF: cellular angiofibroma, MFB:myofibroblastoma, SFT: solitary fibrous tumor, DFSP: dermatofibrosarcomaprotuberans, WLS: well-differentiated liposarcoma, MLS: myxoidliposarcoma. SCL, CAF and MFB specimens were positive for phosphory-lated p38 and p16 in the nucleus and showed decreased expression of β-catenin. In contrast, SFT specimens exhibited decreased phosphorylated p38and p16 expression levels. The others showed various expression levels ofthese markers
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Acknowledgements We thankMr. Yoshihide Oshiro (University of theRyukyus) for his technical assistance.
Compliance with Ethical Standards
Conflict of Interest Statement The authors declare that they have noconflict of interest.
Ethical Approval All procedures performed in studies involving hu-man participants were in accordance with the ethical standards of theinstitutional research committees and with the 1964 Helsinki declarationand its later amendments or comparable ethical standards. For this type ofstudy formal consent is not required.
15. Casini N, Forte IM, Mastrogiovanni G, Pentimalli F, Angelucci A,Festuccia C, Tomei V, Ceccherini E, Di Marzo D, Schenone S,Botta M, Giordano A, Indovina P (2015) SRC family kinase(SFK) inhibition reduces rhabdomyosarcoma cell growth in vitroand in vivo and triggers p38 MAP kinase-mediated differentiation.Oncotarget 6:12421–12435
16. Matsuo T, Shimose S, Kubo T, Fujimori J, Yasunaga Y, Sugita T,Ochi M (2012) Correlation between p38 mitogen-activated proteinkinase and human telomerase reverse transcriptase in sarcomas. JExp Clin Cancer Res 31:5
18. Tong L, Chuang CC, Wu S, Zuo L (2015) Reactive oxygen speciesin redox cancer therapy. Cancer Lett 367:18–25
19. Tsuneki M, Nakamura Y, Kinjo T, Nakanishi R, Arakawa H (2015)Mieap suppresses murine intestinal tumor via its mitochondrialquality control. Sci Rep 5:12472
20. Hoogeboom D, Essers MA, Polderman PE, Voets E, SmitsLM, Burgering BM (2008) Interaction of FOXO with beta-catenin inhibits beta-catenin/T cell factor activity. J BiolChem 283:9224–9230
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